Abstract 2076: Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1
Authors: Naveen K. Mehta, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson
Journal: Cancer Research (2022)

Abstract 4-1BB is a co-stimulatory TNF receptor family member upregulated on activated T cells, where it supports proliferation and enhanced functionality. 4-1BB agonizing monoclonal antibodies have thus far failed to progress beyond early clinical development, either due to hepatic toxicities caused by FcγR-crosslinking (urelumab) or due to low clinical activity (utomilumab). An attractive next-generation approach to 4-1BB agonistic therapeutics are multi-functional biologics that conditionally activate 4-1BB only in the tumor microenvironment, thereby localizing activity and minimizing toxicity. Furthermore, there is a strong rationale to combine 4-1BB agonism with checkpoint inhibition. Opal is a novel multi-specific construct that, in a single molecule, combines (i) a single-chain, trimeric 4-1BB ligand (sc4-1BBL); (ii) an engineered variant of the PD1 extracellular domain (ECD) with affinity-enhancing mutations (mutPD1); and (iii) a serum half-life extension domain. By fusing sc4-1BBL and mutPD1, 4-1BB agonism became conditional on the presence of PDL1 or PDL2. By using the mutPD1 ECD rather than an anti-PDL1 antibody, the specificity of Opal is expanded to PDL2-expressing cancer cells with low to undetectable levels of PDL1. The use of trimeric 4-1BBL is expected to optimally agonize the 4-1BB receptor by engaging its natural binding site. Opal omits a functional Fc gamma domain to avoid the hepatic toxicities associated with urelumab. Opal properties were characterized using biophysical and cellular assays. We showed by surface plasmon resonance spectroscopy that compared to wildtype (wt) PD1, mutPD1 had affinities for PDL1 and PDL2 that were enhanced by &amp;gt;2000-fold and &amp;gt;200-fold, respectively. In a competition ELISA assay, Opal effectively blocked wt PD1 from binding to PDL1 and PDL2. PDL1/2-conditional activation of 4-1BB was shown in two independent assays. First, using 4-1BB reporter cells, 4-1BB activity was enhanced &amp;gt;5x when Opal was pre-complexed with recombinant PDL1-Fc. Second, when activated PBMCs were co-cultured in the presence of HEK293T-PDL1 or -PDL2 cells with serial dilutions of Opal, induction of IFNγ secretion was detectable only in the presence of PDL1 or PDL2. Importantly, variants of Opal with abrogated PD1 or 4-1BBL domains failed to trigger IFNγ production. To evaluate the efficacy and toxicity of Opal in vivo, MC38 tumor cells expressing human PDL1 were inoculated in the flanks of mice expressing human 4-1BB. When tumor-bearing mice were treated IP with Opal at 1 mg/kg, 7/10 mice exhibited durable cures without significant body weight loss. Taken together, the preclinical data suggests that Opal may exhibit single-agent activity in solid tumors by conditionally activating effector cells in the tumor microenvironment via 4-1BB agonism while concurrently blocking the PD1/PDL1/PDL2 axis. Citation Format: Naveen K. Mehta, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson. Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2076.